The present invention relates to a method and compositions for imaging the lymph nodes in a mammal, including a human.
The spread of cancer to regional or distant lymph nodes alters prognosis and treatment. Thus, proper determination of the stage of cancer in a patient requires evaluation of the lymph nodes along the lymphatic chain originating in the cancer. Imaging of lymph nodes is referred to herein as lymphography, and effective lymphography requires that the node be confidently identified, its size determined, and the intranodal anatomy or function be displayed. (The particular techniques disclosed herein may also appropriately be identified as "lymphadenography".) Imaging without direct infusion of contrast agent into the lymph system is referred to as indirect lymphography.
Cancer cells that lodge and grow in lymph nodes may be identified by node enlargement, by altered sieving function, or by altered phagocytosis. Normal lymph nodes range in size from 1-15 mm and can be enlarged by hypertrophy or hyperplasia. Size as a criterion for cancer evaluations is poor unless the nodes are very large and the patient is known to have cancer. Some imaging devices have adequate spatial resolution for sizing lymph nodes, but lack the tissue discrimination to confidently distinguish lymph nodes from other biological structures with similar shapes. These imaging modalities include x-ray, computed tomography, magnetic resonance imaging, and ultrasound. Radioisotope imaging does not have the required spatial resolution.
No contemporary imaging methodology can identify intranodal architecture without a contrast agent. A very few lymphatic channels in the body, usually those of the lower extremity, are large enough to isolate with a surgical procedure and these can be injected with a contrast agent that is carried to their lymph nodes.
Thus, today's conventional technology for imaging lymph nodes utilizes direct infusion of contrast agent into a lymphatic channel. In this procedure, known as lymphangiography, the radiologist directly cannulates a large lymphatic vessel and injects it with a contrast agent, generally an oily iodinated medium that opacifies the sinusoids of a lymph node draining the injected lymphatic vessel. Unfortunately, lymphangiography is only in limited use and does not provide satisfactory results in general. The process requires surgical exposure and identification of the lymph vessel. This is technically difficult, but is an established procedure for lymphatic vessels of the feet. The injection and procedure time is two to three hours and filming is routinely done 24 hours later. Further, the solutions injected tend to image only a few leg, pelvic, and abdominal nodes. It is uncommon to obtain images of any lymph nodes in the chest and neck area, despite the major importance of imaging these nodes in the evaluation and management of lung and breast cancers.
In lymphangiography, the contrast medium seems to be lodged in lymph node sinuses by creating a viscous obstruction that cannot be cleared by hydrostatic forces within the sinusoid. If the lymph node is totally obstructed by cancer cells to begin with or partial obstruction has created collateral channels, the abnormal node will not be visualized and the dye may eventually reach the bloodstream where it will embolize the lungs with adverse effects upon respiratory gas exchange. Some of the major shortcomings of lymphangiography include the fact that direct dissection is required, which is expensive, requires skill and limits repeatability; further, it carries some morbidity. Moreover, the majority of lymphatic drainage beds and their associated lymph nodes are inaccessible; among them are common targets of cancer such as the breast, the testis, the prostate, the cervix, the uterus, the kidneys, the liver, the intestines, and the lungs.
In addition to the danger associated with any prolonged procedure involving surgery, there are other dangers associated with lymphangiography that particularly relate to the contrast agent used in the procedure. If the agent chosen is water soluble or has a particle size that is too small, it will diffuse out of the lymphatic and may also pass through the lymph node too easily. Currently, x-ray lymphangiography utilizes an emulsified ethiodized oil for direct injection into the lymphatic system. This material has a large particle size, is highly viscous, has marginal toxicity, and embolizes the lymph node. In instances where there is an undetected, direct lymphatic-venous connection, ethiodized oils could be directed into the systemic circulation, with harmful and potentially fatal results.
Indirect lymphography with iodine formulations has usually been declared impossible (Mutzel, U.S. Pat. 4,367,216; Hoey, U.S. Pat. No. 4,225,725; Felder, et al., Swiss Patent 615,344) and iodine emulsions are also toxic (Mutzel). On the other hand, Japanese patent publication 42-25413 claimed development of an iodized vegetable fatty oil which was successful with intraperitoneal injection. No details are provided to substantiate the claim; no toxicity data are provided for indirect lymphography nor have there been any reports of other positive experiments since the application date of Sep. 28, 1965. Swiss Patent 615.344 discloses a crystal iodine formulation with a particle size of 1000-5000 nm. They claim that the lymphatic vessels and lymph nodes below the sternum can be regularly visualized after intraperitoneal injection. This is dubious--both as to success and the specific nodes claimed from this injection route, particularly since there is no lymphatic flow downward from the sternum from the peritoneal cavity.
Other emulsions containing iodine have been proposed for lymphography, but have not been adopted for that purpose. See, e.g., Swiss patent No. 615,344 and Japanese published application No. 25413/67. Toxicity problems are one major concern. Also, particle sizes of 1000 nanometers and above are unsuitable for indirect lymphography (with the possible exception of intraperitoneal injection. The peritoneum offers a huge surface and facilitated absorption of most drugs. The lymphatic vessels of the peritoneum are more permeable, especially those of the diaphragm where uptake is facilitated by respiratory motion. Unfortunately, the lymph nodes accessible by this route are few and are of minimal clinical interest. Further, the intraperitoneal route places special limits on the toxicity of the lymphographic agent as direct access to the vascular space is facilitated by the thoracic duct.
Many researchers have experimented with radiolabeled materials as imaging agents for lymphography. There is a relatively rich literature on the development of radioactively tagged agents. Studies show lymphatic uptake and vascular exclusion is optimal for particles of about 40 nm. (See, e.g., Bergquist, et al. "Particle sizing and biokinetics of interstitial lymphoscintigraphic agents," Sem. Nucl. Med 13: 9-19, 1983.) These previous workers have also provided good information on animal models, pharmacokinetics and even human studies. (See also Ege, G. N., "Lymphoscintigraphy-techniques and applications in the management of breast carcinoma," Sem. Nucl. Med 13: 26-41, 1983). Unfortunately, radioactive isotopes trapped in lymph nodes provide poor spatial resolution and make it very difficult to determine details about the size of the nodes or the intranodal architecture.
Au.sup.198 and Ga.sup.67 have some avidity for lymph nodes and tumors, respectively. (See, e.g., Lymphatic Imaging: Lymphography, Computed Tomography and Scintigraphy, 2nd ed., M. Close and S. Wallis, eds., Williams and Wilkins Co., Baltimore, 1985). The former is too energetic for imaging and causes local tissue damage. The latter is interesting, but is only useful for intravenous use and is not suitable for staging of lymph nodes. Recently, protein-specific reagents have been suggested for treatment uurposes. (See, e.g., Weinstein, et al., "Monoclonal antibodies in the lymphatics: toward the diagnosis and therapy of metastases," Science 218: 1334-1337, 1982). At the moment, however, spatial resolution of isotope imaging devices precludes capitalizing on the selective distribution of tagged materials within lymph nodes.
In general, soluble and relatively small molecules such as albumin (&lt;5 nM) are either better absorbed from the interstitial space into blood than into lymph or they are poorly retained by lymph nodes so that they are ineffective for imaging intranodal architecture.
We consider larger molecules to be particles and those up to 1 micron are called colloids while above 1 micron the particles are called suspensions. Microaggregated albumin fits in the former category while macroaggregated albumin is in the latter. Radiolabelled colloids have been of great interest for indirect lymphography, despite the low spatial resolution of current cameras. However, the methods for measuring the size of candidate agents are poor. Bergquist et al (Sem. Nucl. Med. 1983;13:9) list 9 different techniques for measuring radiocolloid particle size. None is totally satisfactory and many colloid preparations also have a wide range of particle sizes. A bimodal distribution in a particular preparation would invalidate a method that measures average size. Most preparations are better characterized by a complete distribution or histogram.
If the particles are rigid, then sizing is easier. But if the particles are deformable, then sizing is more difficult. Most techniques measure size in vitro, but size may increase or decrease in vivo (Bergquist). Colloidal particles are usually coated with a stabilizer prior to administration and are also coated (opsonized) in vivo. The effective particle size includes the coat as well as the core, so this complicates the measurement. For imaging uses, the active contrast agent is usually limited to the core material.
The particles enter the lymphatics from the interstitium through gaps between lymphatic endothelial cells or by transcellular endo-exocytosis. The gaps change in calibre with physiologic or pathologic conditions. Entry of the particle into the gap is believed to be a hit-or-miss affair and should be weakly related to particle size at dimensions less than the size of the gap. On average, smaller particles (10-50 nM) are more likely to enter than larger particles. However, larger particles usually carry more imageable material and are more effective per particle in altering the image intensity. Thus, predicting imaging efficacy for a particular formulation is complicated. As particles approach 1000 nM, their uptake into lymphatics is so poor that they become ineffective. Very large particles in the interstitial space must be carried away by phagocytes or reduced in size by local processes.
Once particles reach the lymphatic space, a functional lymph node can very efficiently remove them, even if they are as large as a few thousand nanometers. This process requires particle coating, adhesion, and phagocytosis. Cancer deposits in the lymph node destroy lymph node sinusoids and cancer cells have little or no phagocytic capacity. For both these reasons, cancerous regions accumulate particles poorly.
Virtually all particles--and the list is long and varied--selectively target normal parts of the node from the lymph in which they are carried. The influence of "sick phagocytes" upon sequestering particles in lymph nodes is unknown, but most processes causing hyperplasia or hypertrophy create functioning phagocytes and these nodes, though large, accumulate particles.
Ethiodol--the standard lymphangiographic agent--is poorly phagocytosed and acts by sinusoidal blockade. This accounts for its spotty distribution creating a grainy appearance in normal nodes at moderate magnification. The germinal follicles in lymph nodes have few sinusoids and also few functioning macrophages. Both Ethiodol and particles will be sparsely accumulated in these regions.
A lymph node totally replaced by other cells, usually cancer, will receive no lymph but does continue to receive blood. Neither direct nor indirect lymphography will be effective in defining intranodal architecture in this circumstance. It may still be possible to target such nodes with intravascular agents.
Clearly, despite the elegant compartmental models of Strand et al. (J. Nucl. Med. 1979;20:1038), most attempts to evaluate indirect lymphography agents rely heavily on actual animal experimentation. Mikheev (Atomic Energy Review 1976;14:1) writes, "As the measurement of colloid particle size presents great difficulties and the behavior of colloidal solutions in the body is only indirectly related to particle size, it is more useful to control colloidal solutions by biological studies in laboratory animals." On the other hand, it would be much easier to screen proposed agents in vitro were size distribution and imaging efficacy methods available and accurate. Such data would dramatically reduce the burden for animal studies and would enable the investigator to identify anomalous responses that might aid in further understanding of this complex biological process.
There is at least one reported attempt at indirect lymphography through injection of radiopaque perfluorocarbons subcutaneously into the peritoneal or pleural spaces or into the lung parenchyma. However, animal tests involving injection of both neat and emulsified radiopaque perfluorocarbon failed to produce clinically useful information. D. Long, et al., Radiology 133: 71-76 (1979). Instead, radiopacification of lymph nodes with emulsions was sporadic and was observed only in one animal eight months following administration of the perfluorocarbon. This outcome is too inconstant and too delayed for clinical applications.
Other attempts to perform direct or indirect lymphography have utilized dimers of iodinated water soluble agents. The dimer increases molecular size, and reduces diffusion from the lymphatic to some degree; however, these small soluble agents appear to provide only transient opacification of the lymph node.
Accordingly, there is a need for a lymphographic technique and contrast agent that permits imaging of lymph nodes in any desired area of the body within a reasonable time period. Such imaging would identify location, size, and internal architecture of regional lymph nodes of interest and would permit differentiation between lymph node enlargement due to hypertrophy and hyperplasia of normal node constituents, on the one hand, and neoplasia, on the other hand.
Moreover, there is a need for a lymphographic procedure that minimizes procedure time and patient discomfort, while reducing the dangers of the procedure.
These and other objectives are met by the present invention.